Curr. Issues Mol. Biol. 14: 9-26. Ribonucleotide Reductase as a Target to ControlOnline Apicomplexan journal at http://www.cimb.org Diseases 9 Ribonucleotide Reductase as a Target to Control Apicomplexan Diseases James B. Munro and Joana C. Silva* hosts/vectors and transition between life cycle stages is dependent upon a diverse array of environmental cues. Department of Microbiology and Immunology and Institute The biological characteristics that differentiate for Genome Sciences, University of Maryland School of apicomplexans from their vertebrate hosts have often been Medicine, Baltimore, MD 21201, USA considered optimal targets of new therapeutics to control these eukaryotic pathogens. Alternatively, essential and strongly conserved proteins can be targeted, provided that Abstract they differ from their vertebrate homologs in such a way that Malaria is caused by species in the apicomplexan genus minimizes potential cross-reaction and toxicity. Plasmodium, which infect hundreds of millions of people The enzyme ribonucleotide reductase (RNR) is one each year and kill close to one million. While malaria is such example. RNR utilizes free radical chemistry to catalyze the most notorious of the apicomplexan-caused diseases, the reduction of ribonucleotides to deoxyribonucleotides other members of the eukaryotic phylum Apicomplexa (Thelander and Reichard, 1979; Reichard, 1988). It provides are responsible for additional, albeit less well-known, the only de novo means of generating the essential building diseases in humans, economically important livestock, and blocks for DNA replication and repair across all domains a variety of other vertebrates. Diseases such as babesiosis of life and, as such, it is the rate-limiting step in DNA (hemolytic anemia), theileriosis and East Coast Fever, synthesis (Jordan and Reichard, 1998; Lundin et al., 2009). cryptosporidiosis, and toxoplasmosis are caused by the Additionally, RNR is critical for maintaining a balanced pool apicomplexans Babesia, Theileria, Cryptosporidium and of DNA precursors during chromosome replication (Herrick Toxoplasma, respectively. In addition to the loss of human and Sclavi, 2007). An unbalanced deoxyribonucleotide life, these diseases are responsible for losses of billions triphosphate pool may lead to an increase in mutation and of dollars annually. Hence, the research into new drug disease (Lin and Elford, 1980; Reichard, 1988; Chabes et targets remains a high priority. Ribonucleotide reductase al., 2003; Wheeler et al., 2005; Gon et al., 2006; Mathews, (RNR) is an essential enzyme found in all domains of 2006; Kumar et al., 2010). life. It is the only means by which de novo synthesis of Here we review the chemotherapeutic methods used to deoxyribonucleotides occurs, without which DNA replication inhibit the essential enzyme RNR, with particular emphasis and repair cannot proceed. RNR has long been the target of on the novel RNR R2_e2 subunit recently identifed in antiviral, antibacterial and anti-cancer therapeutics. Herein, apicomplexan parasites (Munro et al., submitted) and on the we review the chemotherapeutic methods used to inhibit malaria-causing genus Plasmodium. The R2_e2 subunit is RNR, with particular emphasis on the role of RNR inhibition unique to the Apicomplexa and as such, it can potentially be in Apicomplexa, and in light of the novel RNR R2_e2 subunit used to specifcally target apicomplexan pathogens. recently identifed in apicomplexan parasites. Apicomplexan parasites are responsible for devastating Introduction infectious diseases The Apicomplexa are a group of single-celled, eukaryotic The phylum Apicomplexa consists of more than 4,000 organisms that, together with the ciliates and dinofagelates, described species (Levine, 1988), many of which are of form the major lineages in the Alveolates. All Apicomplexa, medical, agricultural, and economic importance and whose save the predatory fagellates Colpodellida, are pathogenetic, adverse impact on human society cannot be overstated. obligate, intracellular parasites (Adl et al., 2005; Morrison, Among the most notorious are Plasmodium, Babesia, 2009). They are characterized by the presence of an apical Theileria, Cryptosporidium, and Toxoplasma the causative complex, a structure involved in host-cell invasion, which is agents of malaria, babesiosis, theileriosis and East Coast located at the anterior end of the cell. Most apicomplexans fever, cryptosporidiosis, and toxoplasmosis, respectively. also possess a specialized organelle called the apicoplast, They are responsible for causing millions of human deaths a secondary endosymbiotic plastid believed to be of red and billions of dollars in productivity and material losses algal origin (Blanchard and Hicks, 1999; Fast et al., 2001; each year (Sachs and Malaney, 2002; Corso et al., 2003; Janouškovec et al., 2010). Within the apicoplast occur Rowe et al., 2006; Spielman, 2009). Currently, fve species processes essential for the parasite's survival, such as of Plasmodium are known to cause malaria in humans, P. heme and lipid biosynthesis. Another defning characteristic falciparum, P. knowlesi, P. malariae, P. ovale, and P. vivax of apicomplexans is their inability to synthesize purine rings (Rougemont et al., 2004; Singh et al., 2004; Cox-Singh et de novo and hence their need to salvage exogenous purines al., 2008), of which P. falciparum is the most deadly and via a variety of different pathways (Booden and Hull, 1973; P. vivax the most geographically widespread. The life cycle Chaudhary et al., 2004; de Koning et al., 2005; Cassera of Plasmodium alternates between a vertebrate host and et al., 2008; Madrid et al., 2008). These organisms have mosquito vector and involves four major developmental complex (indirect) life cycles, and they often exploit multiple stages in the vertebrate host: sporozoites, merozoites, trophozoites, and gametocytes (Bledsoe, 2005; Brown and Catteruccia, 2006). *Corresponding author: [email protected] Horizon Scientifc Press. http://www.horizonpress.com 10 Munro and Silva Prioritization of apicomplexan drug targets been proposed as a means to combat the emergence of There is currently no fully effcacious vaccine on the market drug resistance (Durand et al., 2008). Genes with high rates against any apicomplexan species and drug treatment is of nonsynonymous changes have been associated with the method of choice in the management of apicomplexan drug resistance in P. vivax (Dharia et al., 2010), and may be diseases. Modern approaches to drug design, made responsible for vaccine evasion in P. falciparum (Takala and possible with the advent of genome sequencing, emphasize Plowe, 2009). In contrast, evolutionary patterning focuses defning and targeting metabolic and molecular differences on fnding and targeting protein residues that are under between host and parasite to avoid host side effects (Croft, strong purifying selection, which will in principle reduce the 1997). This may be achieved with the selective targeting of instances of drug resistance mutations. parasite-specifc enzymes or by targeting those which are The tremendous health burden imposed by malaria highly divergent or have distinct binding sites and are thus has made Plasmodium the primary target for many of these suffciently different to be selectively targeted (Coombs, approaches. Despite a diversifed arsenal of potential tools 1999; Cerqueira et al., 2007). Also important is that the to combat malarial infection, multiple drug resistance to protein target be essential to the growth, reproduction, or existing anti-malarial compounds is becoming increasingly survival of the parasite. Finally, knowledge of the gene common in Plasmodium (Greenwood and Mutabingwa, expression pattern of potential target proteins is necessary to 2002; Anderson, 2009; Bustamante et al., 2009; Takala and link drug administration with the critical and relevant stages Plowe, 2009). A case in point is the antifolate drugs used to of the pathogen's life cycle. This is particularly pertinent to treat malaria. Antifolate drugs bind enzymes necessary for organisms characterized by differentially expressed, stage- folate biosynthesis, thus targeting essential precursors for specifc, and often stage-unique gene expression, such as purine and pyrimidine synthesis. Antifolates have been used apicomplexans (Coulson et al., 2004). It has been suggested against Plasmodium with success, but resistance to these that targeting the intraerythrocytic life stages of Plasmodium drugs has become widespread (Gregson and Plowe, 2005; (the ring forms, trophozoites, schizonts, and merozoites), Mkulama et al., 2008; Sridaran et al., 2010). Currently, the when clinical symptoms are manifested, is of particular primary treatment for malaria is based on artemisinin, which interest (Yeh and Altman, 2006). However, targeting multiple is administered in combination with other drugs in order to life stages, for example controlling both the liver and blood prevent, or delay, the onset of resistance. However, there stages of Plasmodium, may be more conducive to the are clear indications that artemisinin resistance is emerging ultimate goal of disease eradication (Alonso et al., 2011). (Chrubasik and Jacobson, 2010; Dondorp et al., 2010; Enserink, 2010; Fidock, 2010). Therefore, the development Current control strategies and challenges of new chemotherapeutic and prophylactic antimalarial Drug targets for control of apicomplexans have focused on drugs and vaccines remains a priority (Greenwood and parasite